The wild-type (WT) human pol β cDNA was cloned from a human cDNA library. R137Q substitution was obtained by site-directed mutagenesis using the primers shown in Table 1. WT and R137Q genes were inserted into the pET28b vector and expressed in Escherichia coli strain BL21. Expressed proteins were purified as previously described (40). Table 1.

Oligonuleotide sequences and their applications in this study

The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see Table 1 and Figure 1A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-³²P]-dCTP and 0–10 ng WT or R137Q Pol β. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer. The left portion of reaction product was then stopped by addition of equal volumes of gel loading buffer (90% formamide dye, 3 M EDTA, 0.02% bromophenol blue and 0.02% xylene cyanol), heated (5 min, 95°C), separated by 15% Polyacrylamide gel electrophoresis (PAGE) containing 8 M urea and visualized by autoradiography. Figure 1.

The Pol β polymorphism R137Q is defective in polymerase activity. (A) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP-³²P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. (B) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. (C) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by ³²P as shown in figure. (D) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.

Gel shift and ELISA-based isothermal absorption assays were used to compare the DNA-binding affinity between WT and R137Q Pol β. For gel shift assays, various concentrations of Pol β protein (0.1–1000 nM) were incubated (15 min, room temperature) with 0.1 nM radio-labeled Pol-GAP substrate in a buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl₂, 10% glycerol and 0.1% NP-40. Samples were run on a 5% native polyacrylamide gel and visualized by autoradiography. For ELISA-based affinity assays, biotin-labeled Pol-GAP DNA substrate (1 pmol) was immobilized on a streptavidin-coated 96-well Enzyme-Linked ImmunoSorbent Assay (ELISA) plate and washed three times with binding buffer, followed by the addition of 0–1 µg WT or R137Q Pol β and incubated (overnight, 4°C). Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313). Color was developed by adding tetramethyl benzidine (TMB) and stopped by addition of 1 N HCl. The OD₄₅₀ was read on a microplate reader.

The dRP lyase assay using purified Pol β protein was performed as described previously (41). To prepare the dRP lyase substrate, the DNA substrate Pol β-U was labeled with (γ-³²P)-ATP at the 5′-end on the U-containing oligonucleotide, followed by treatment with uracil-DNA glycosylase (UDG) and APE1. The incised AP-site-containing DNA was then incubated (20 min, 37°C) with Pol β (0–10 ng) in a buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 20 mM KCl and 2 mM DTT, stabilized by addition of 2 M sodium borohydride to a final concentration of 340 mM, followed by incubation on ice (30 min). Formamide-containing gel loading buffer was then added and reaction products were resolved on 15% polyacrylamide gels containing 8 M urea and visualized by autoradiography.

The BER assay was performed as described previously (22,42,43). Complete repair reactions were carried out in 20 µl of reaction buffer [40 mM HEPES–KOH (pH 7.8), 70 mM KCI, 7 mM MgCl₂, 1 mM dithiothreitol, 0.5 mM EDTA, 2 mM ATP, 50 µM each of dATP, dTTP and dGTP, and 8 µM 2 µCi (α-³²P)-dCTP]. For short-patch reconstitution with purified proteins, UDG (8 ng), APE1 (2 ng), Ligase IIIα (20 ng) and various amounts of Pol β (0–5 ng), were mixed and incubated with the SP-BER substrate Pol β-U (Table 1). For LP BER, Pol β-F substrate (Table 1) was incubated with the mixture of APE1 (2 ng), Pol β (0–5 ng), FEN1 (2 ng), and Ligase I (20 ng). Note that in the LP-BER substrate, radio-labeled dCTP can only be incorporated in the second position next to the damaged base. For cell extract reconstitution, SP- or LP-BER DNA substrate was incubated with the total cell extracts (0–5 µg). Reactions (30 min, 37°C) were then stopped by adding an equal volume of the gel loading buffer and visualized by autoradiography.

WT and pol β null (pol β^−/−) mouse embryonic fibroblast (MEF) cells were described previously (15). Cells were grown in Dulbecco's; Modified Eagle Medium (DMEM) medium supplemented with 10% FBS and 100 units each of penicillin, streptomycin and hygromycin. The pol β^−/−/WT and pol β^−/−/R137Q stable cell lines were generated by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Selection was carried out using 100 µg/ml of G418.

Cell extracts were made according to Biade et al. (43). Briefly, cells were cultured in 10 cm² dishes, incubated overnight to reach a mid-exponential growth phase, washed three times with ice-cold PBS and resuspended at 10⁶ cells/20 µl in buffer I [10 mM Tris–HCl (pH 7.8) and 200 mM KCl]. After the addition of an equal volume of Buffer II [10 mM Tris–HCl (pH 7.8), 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% NP-40, 2 mM dithiothreitol, 0.5 mM PMSF and protease inhibitor cocktail (Roche)], the cell suspension was rocked (1 h, 4°C), centrifuged (10 min, 16 000g) and the supernatant recovered and stored (−80°C). Protein concentrations were determined by Bradford protein assay.

To determine the interaction between R137Q and proteins in the BER complex, His-tagged Pol β protein (20 µg) was mixed with cell extract prepared from the pol β^−/− MEF cell line (50 µg) in 150 µl binding buffer [50 mM Tris–HCl (pH 7.6), 75 mM KCl, 1 mM DTT, 10 mM imidazole], incubated (60 min, on ice) and 15 µl of pre-equilibrated nickel beads were added to the mixture which was once again incubated (1 h, on ice, with constant shaking). Beads were then washed five times with the binding buffer, suspended in SDS–PAGE sample buffer (50 µl) (31), boiled (5 min, 95°C) and the supernatant subjected to SDS–PAGE followed by western blotting with antibodies against APE1 (Genetex, GTX70132), FEN1 (Genetex, GTX70186), PCNA (Genetex, GTX70286), Ligase IIIα (Genetex, GTX70144), Ligase I (Genetex, GTX70142) and XRCC1 (Genetex, GTX70262). To confirm the interaction between Pol β and PCNA, purified PCNA was immobilized on Sepharose 4B, as previously described (40); PCNA coated beads were then mixed with purified WT or R137Q Pol β. After incubation (60 min, on ice) and washing with binding buffer, Sepharose 4B beads were boiled in SDS–PAGE sample buffer and the supernatant analyzed by western blotting with anti-Pol β and PCNA antibodies.

DNA damage assay was carried out as described (44). Briefly, monolayer cells were treated (1 h) with MMS at different concentrations, collected and genomic DNA was extracted by using DNAzol reagent (Invitrogen). Extracted DNA was then coated onto ELISA plates and detected by AP-site specific reagent, Aldehyde-Reactive Probe (ARP) (Invitrogen), followed by detection with HRP-conjugated streptavidin and TMB. After stopping by 1N HCl, OD₄₅₀ was measured on a microplate reader.

Sensitivity to MMS and MNU was determined by HeLa cell growth inhibition assay. HeLa cells were seeded (1500/well), incubated (overnight, 37°C). Treated (1 h, 37°C) with multiple dilutions of MMS, washed in a fresh medium (DMEM containing 10% fetal bovine serum) and incubated (72 h) under normal growth conditions (37°C, 5% CO₂). The number of viable cells was determined by the CellTiter 96 AQueous one-solution cell proliferation assay (Promega). At least four replicas for each clone were averaged. Data are expressed as the percentage of growth relative to untreated controls.

The cell cycle distribution and apoptosis of stably complemented cell lines were analyzed by Fluorescence Activated Cell Sorting (FACS) analysis, as previously described (45). Harvested cells (10 cm² dish) were fixed with pre-chilled 70% ethanol, stained with propidium iodide (PI; Sigma) and analyzed by flow cytometry at the City of Hope Flow Cytometry Core Facility.

The Pol β polymorphism (rs12678588) encoding an Arg137 to Gln substitution was initially identified by the Human Genome Project and was confirmed by Mohrenweiser et al. (46). This polymorphism has been identified in Caucasians, Africans and Asians with rare occurrence frequency (National Center for Biotechnology Information database and reference 38). However, the extent of the impact of this polymorphism on disease development, especially carcinogenesis, has not been fully elucidated. To determine the effects of the R137Q Pol β variant on biochemical activities and/or biological functions, we first expressed and purified WT and R137Q human Pol β proteins, and then assayed the primer extension, dRP lyase and substrate-binding activity in vitro, using synthetic DNA substrates (Table 1). We found that the R137Q variant had ∼30% primer extension activity compared to the WT enzyme (Figure 1A and B). However, no differences were observed in the R137Q variant for the DNA-binding activity or dRP lyase activity (Figures 1C, D and 2). These results were consistent with the fact that R137Q occurred at the 31-kDa domain, the polymerase catalytic domain, while the 8-kDa domain was responsible for dRP lyase and DNA-binding activity. Figure 2.

dRP lyase activity assay of Pol β. (A) Schematic representation of the reactions generating a 5′-dRP-group-containing DNA substrate. Treatment of Pol β-U (with ³²P labeled at 3′end) with UDG generates an AP-containing DNA, which then was incised at the nucleotide 5′ to the AP site by APE1, resulting the 5′-dRP-containing substrate (20 nt + dRP). Removal of the 5′-dRP residue by dRP lyase results in a 3′-³²P labeled 20 nt DNA fragment. Asterisk represents ³²P labeling. (B) 5′-dRP lyase activity assay. (C) Quantification of the 5′-dRP lyase activities shown in (B).

We then investigated the effects of R137Q substitution on protein–protein interactions. Extracts of pol β^−/− MEF were incubated with purified WT or R137Q proteins (His-tagged) prior to immunoprecipitation of proteins involved in the BER pathway and detection by corresponding antibodies (Figure 3). Six proteins (APE1, FEN1, PCNA, XRCC1, Ligase I and Ligase IIIα) that interact with Pol β and are involved in either the SP- or LP-BER pathways were investigated. The amount of APE1, FEN1, XRCC1, Ligase 1 or Ligase IIIα that co-pulled down with R137Q was similar to that with the WT enzyme. However, there was a dramatic decrease in levels of R137Q-bound PCNA compared with WT Pol β-bound PCNA (Figure 3A, panel 2). To rule out the possibility that the His tag itself is responsible for the observed binding, we included His-tagged glutathione S-transferase (GST) as a control. In this case, none of the tested proteins binds to His-tag or GST. To further confirm this observation, we immobilized purified PCNA on Sepharose 4B beads and incubated them with purified WT or R137Q Pol β. Western blot of Sepharose bead-bound proteins showed that WT Pol β but not R137Q was efficiently pulled down by PCNA (Figure 3B), suggesting that the Arg137 to Gln substitution impaired the interaction between Pol β and PCNA. To quantitatively evaluate the impact of the R137Q substitution on the Pol β/PCNA interaction, we determined the relative amount of WT Pol β or R137Q-bound PCNA by ELISA. Our data suggested that levels of WT Pol β-bound PCNA were 1.5- to 4-fold higher than those of R137Q-bound PCNA (Figure 3C). Figure 3.

The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β null MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.

The impaired biochemical functions of the R137Q variant suggested that R137Q likely affected BER function. To test this hypothesis, SP- and LP-BER were assayed using purified proteins. Uracil-containing substrate (Pol β-U) and tetrahydrofuran-containing substrate (Pol β-F) were used for SP- and LP-BER substrates, respectively (Table 1). Cleavage of the uracil or Tetrahydrofuran (THF) lesion by the concerted action of UDG and APE1 resulted in a nicked DNA duplex, and incorporation of ³²P-dCTP and other deoxynucleotides produced 20–30 nt non-ligated intermediates visible to the phosphorimager (Figure 4A and B). Further processing of this intermediate structure generated a fully repaired product of 40 nt. We observed that the uracil or THF lesions were efficiently repaired in the presence of WT Pol β but not the R137Q variant (Figure 4A and B). The reduced repair efficiency by R137Q was likely due to its low-polymerase activity. Consistent with the polymerase activity assays (Figure 1A), only a small amount of ³²P-dCTP was incorporated into the DNA substrate by R137Q in reconstituted SP- and LP-BER reactions (Figure 4A and B). To validate that the R137Q variant lead to low-BER efficiency, we expressed human WT and R137Q Pol β in mouse MEF of pol β knockout genetic background. Cells expressing similar level of Pol β were selected (Figure 5A). Nuclear extracts (NEs) from these cell lines were prepared and their BER efficiency assayed. We found that WT NE efficiently repaired the uracil or THF lesion, resulting in a 40 nt band, whereas the repair efficiency by R137Q NE was only ∼10 and 20% for SP- and LP-BER, respectively (Figure 5B and C). Figure 4.

R137Q significantly reduces BER efficiency. (A) SP-BER reconstitution with purified WT and R137Q Pol β. (B) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.

Our observation that R137Q Pol β caused defective BER led us to propose that cells expressing the R137Q variant would be sensitive to DNA base damaging agents. To test this hypothesis, MEF cells of WT, pol β knockout (pol β^−/−) and pol β^−/− expressing human WT or R137Q Pol β (designated as pol β^−/−/WT or pol^−/−/R137Q) were treated with MMS. Consistent with a previous report (15), we found that deletion of pol β caused cells to accumulate MMS-induced DNA damage at MMS concentrations ≥1 mM (Figure 6A) and to become sensitive to the MMS and MNU (Figure 6B–D). While human WT Pol β fully complemented the function of its mouse homolog, R137Q only partially complemented it. FACS data showed that apoptotic cells of pol β knock out or R137Q genetic background were considerably higher than the WT background. Additionally, there was a 5-fold increase in MMS-induced apoptotic R137Q cells compared with WT (3.1% versus 0.6%) (Figure 6B). MMS consistently had a stronger inhibitory effect on pol β^−/−-R137Q cell growth relative to WT or pol β^−/−/WT cells. Following MMS or MNU treatment, the ratio of MMS or MNU treated/untreated pol β^−/−-R137Q cells was <10%, whereas that for WT or pol β^−/−-WT cells was >90% (Figure 6C and D). Figure 6.

Cells harboring polymorphism R137Q are sensitive to DNA damage stress. (A) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). (B) Effect of MMS on induction of apoptosis in complemented pol β^−/− MEFs. Pol β^−/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β^−/−, pol β^−/−/WT and pol β^−/−/R137Q MEF cells were treated (1 h) with MMS (C) and MNU (D) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β^−/−/WT, filled triangles; pol β^−/−, filled circles; pol β^−/−/R137Q, open squares.